Xylanases are glycosyl hydrolases which hydrolyse β-1,4-linked xylopyranoside chains. Xylanases have been found in at least a hundred different organisms. Together with other glycosyl hydrolases they form a superfamily which includes more than 40 different enzyme families (Henrissat and Bairoch, 1993). Family 11 (previously G) xylanases are defined by the similarities in their gene sequences, protein structures, and catalytic mechanisms. Common features for the members of this family are high genetic homology, a size of about 20 kDa, and a double displacement catalytic mechanism (Tenkanen et al., 1992; Wakarchuk et al., 1994).
The family 11 xylanases mainly consist of β-strands which form two large β-sheets, and of one α-helix. These form a structure that resembles a partly-closed right hand, wherein the β-sheets are called A- and B-sheet. (Törrönen & Rouvinen, 1997). The family 11 xylanases have special interest in industrial applications, because their structure is stable, and they are not susceptible to protease activity. In addition, xylanases can be produced economically on an industrial scale. Trichoderma reesei is known to produce three different xylanases of which xylanases I and II (XynI and XynII) are the best characterized (Tenkanen et al., 1992). XynI has a size of 19 kDa, and compared to XynII it has low isoelectric point and pH optimum (pI 5.5, pH 3-4). XynII has a size of 20 kDa and it has a pI of 9.0 and a pH optimum of 5.0-5.5 (Törrönen and Rouvinen, 1995).
The most important industrial applications of xylanases are pulp bleaching, modification of textile fibres, and biomass modification to improve its digestion in animal feeding (Prade, 1996). A common nominator in all these applications is the extreme conditions which face the enzyme. High temperatures, and pH which substantially differs from the optimal pH of many xylanases decrease the effective utility of the presently available xylanases in industrial applications.
In feed applications the enzyme faces high temperature conditions for a short time (e.g. 2-5 min at 90° C.) during feed preparation. However, the actual catalytic activity of the enzyme is needed at lower temperatures (e.g. ˜37° C.). Consequently, the enzyme should not be inactivated irreversibly at high temperatures, while it has to be active at relatively low temperatures.
In pulp bleaching the material coming from alkaline wash has a high temperature (>80° C.) and pH (>10). None of the commercially available xylanases survives these conditions. The pulp must be cooled and the alkaline pH neutralized in order to treat the pulp with presently available xylanases. This means increased costs. Protein engineering has been used—sometimes successively—to stabilise xylanases to resist the denaturing effect of the high temperature and pH.
Several thermostable, alkaliphilic and acidophilic xylanases have been found and cloned from thermophilic organisms (Bodie et al., 1995; Fukunaga et al., 1998). However, production of economical quantities of these enzymes has in most cases proved to be difficult. Therefore the T. reesei xylanase II, which is not as such thermostable, is in industrial use because it can be produced at low cost in large quantities. As an alternative for isolating new xylanases, or developing production processes, one can envisage engineering the presently used xylanases to be more stable in extreme conditions.
The stability of Bacillus circulans xylanase has been improved by disulfide bridges, by binding the N-terminus of the protein to the C-terminus and the N-terminal part of the α-helix to the neighbouring β-strand (Wakarchuk et al., 1994). Also Campbell et al. (1995) have modified Bacillus circulans xylanase by inter- and intramolecular disulfide bonds in order to increase thermostability. On the other hand, the stability of T. reesei xylanase II has been improved by changing the N-terminal region to a respective part of a thermophilic xylanase (Sung et al., 1998). In addition to the improved thermostability, the activity range of the enzyme was broadened in alkaline pH. Single point mutations have also been used to increase the stability of Bacillus pumilus xylanase (Arase et al., 1993). The influence of mutagenesis on stability has been studied on many other enzymes. By comparing the structures of thermophilic and mesophilic enzymes plenty of information has been obtained (Vogt et al., 1997). Structural information of thermophilic xylanases has also given information about factors influencing the thermostability of xylanases (Gruber et al., 1998; Harris et al., 1997).